Diagnosis of viral infections can be made by methods of detecting viruses or virus-related components (proteins and nucleic acids), and methods of determining specific antibodies produced by living organisms due to viral infection. In the infection of the hepatitis B virus (HBV), diagnostic markers such as HBs antigen and antibody, HBc antibody, HBe antigen and antibody, and HBV-DNA have been introduced in laboratory tests.
Generally, the presence of HBV infection can be known by the HBs antigen or the HBc antibody. Not only for infectivity of HBV but as indices for judging pathology and prognosis of HBV carriers, and therapeutic effects, determinations of HBe antigen and antibody and the amount of HBV-DNA have been used. Due to its simple and inexpensive procedures, immunological measurement of HBe antigen and antibody, among them, has been most widely used. In precore mutants that cannot produce or secrete HBe antigen, however, there are cases in which the propagation of HBV is active, infectivity and immunogenicity are potent, and pathology is active though HBe antigen is negative. On the other hand, regardless of whether they are wild types or mutants, the measurement of the amount of HBV-DNA that reflects the propagation and replication state of HBV is important, but it has problems that the procedures of pre-treatment and measurement are complicated, and lack reproducibility and stability.
Recently, a method of determining HBV core-related antigens was developed that is thought to reflect the propagation and replication state of HBV despite the presence of HBV core-related antibodies (HBC antigen and HBe antibody) or the appearance of mutants. Kimura et al. (Journal of Clinical Microbiology, 40:439-445, 2002) have developed an immunoassay that simultaneously detects HBc antigen and HBe antigen in the serum by a simple pre-treatment using a monoclonal antibody having a specificity for the HBV core-related antigens (HBV precore/core gene products containing HBc antigen and HBe antigen), and have demonstrated that it has a correlation with the NAT testing method that detects the virus genome.
According to the literature so far, the HBV precore/core gene encodes a protein comprising a full-length of 212 amino acid residues (amino acid Nos. −29 to 183, when the first amino acid of the HBc antigen was set as 1. The same holds true hereinafter), and has a signal peptide of 19 residues at the N-terminal thereof. The signal peptide comprises hydrophobic amino acid residues and functions as a signal sequence for the protein secreted to bind to the membrane. Here, it represents a sequence comprising amino acids at −29 to −11.
The precore protein, when translated, passes through the endoplasmic reticulum membrane and, after cleaving the 19-residue signal peptide, the C-terminal nucleic acid-binding domain is cleaved to become the HBe antigen (amino acid Nos. −10 to 149), which is secreted into the blood. This HBe antigen was conventionally thought to occur in association with serum proteins such as albumin and γ-globulin in the blood, and it had not been reported to form HBV particles. There are also some reports that HBe antigen is involved in the maintenance of immunological tolerance, suppression of virus replication etc., but it is not indispensable for HBV replication, and there is much to be elucidated on its biological role.
HBV precore/core gene has the second transcription initiation signal, and a product transcribed and translated from this signal turns into the HBc antigen (amino acid Nos. 1-183), which, while incorporating HBV pregenome RNA, forms a virus capsid and is coated with an outer coat (envelope) comprising HBs antigen and, then, is extracellularly released as complete virus particles (Dane particles). It has been reported that when the amino acids at positions 1-144 among the HBc antigen are present, core particles are formed and the molecule forming the core particles of the infectious virus are believed to be comprised of a protein having an amino acid sequence at positions 1-183.
Though HBc antigen and HBe antigen are proteins that share the majority of the sequences, they have significantly different properties. Significantly responsible for this difference is a cysteine residue of the amino acid at position −7 of the HBe antigen. This cysteine, in combination with the cysteine residue at amino acid position 61 in the same molecule, forms a disulfide bond and acquires the properties of e antigen (M. Nassal and A. Rieger, Journal of Virology, 67: 4307-4315, 1993). The fact that when, for example, the cysteine at position −7 is replaced with another amino acid to prevent the disulfide bond from forming, the conformation of HBe antigen is not taken also indicates the significance of this disulfide bond.
On the other hand, as HBc antigen is transcribed and translated from the amino acid at position 1, it does not have the cysteine residue at position −7 and thus takes the HBc conformation, and two molecules of HBc protein turn into a homodimer by forming a disulfide bond between the cysteine residues at amino acid position 61, thereby forming a virus capsid (core particles).
The HBV core-related antigens occurring in the serum of HBV patients are roughly divided into the HBc antigen (amino acid Nos. 1-183) that constitutes the infectious Dane particle and the secretary HBe antigen (amino acid Nos. −10 to 149) that is almost the same as the HBc antigen in terms of amino acid sequence but has an antigenicity different from that of the HBc antigen. However, in addition to the above, the presence of pre-HBe antigen (amino acid Nos. −29 to 149) in the blood that exhibits a HBe antigenicity as a minor molecule has been demonstrated by Takahashi et al. (Journal of Immunology, 147, 3156-3160, 1991).
They separated the pooled sera by centrifugation, and purified from the supernatant a protein having a HBe antigenicity using an affinity column of anti-HBe monoclonal antibody and sequenced it. As described above, the HBe antigen protein is thought to occur in the blood without forming virus particles. They also have decided the HBe antigen sequence that was purified without peeling off (disrupting) the HBs antigen which is a coat protein of HBV or without disrupting virus particles and core particles, and the HBe antigen having the signal sequence (amino acid Nos. −29 to 149) demonstrated by them is believed to occur in a free state in the serum.
On the other hand, it has been reported that, among Dane particles, there are some empty particles containing no HBV-DNA (SAKAMOTO et al., Laboratory Investigation, 48, 678-682, 1983) or replication-defective deficient particles containing shorter-than-usual HBV-DNA that underwent splicing (TERRE et al., Journal of Virology, 65, 5539-5543, 1991), but the HBV protein constituting the particle has not been analyzed in detail.
Thus, biochemical characterization of the HBV core-related antigens analyzed in the above literature cannot be considered sufficient, and the form of the HBV core-related antigens as virus markers in the blood that is thought to reflect the state of propagation and/or replication of HBV has not been resolved yet.